A thermodynamic model for ligand-induced biphasic thermal protein denaturation, which derives from experimental studies of the thermally induced denaturation of human albumin in the absence and presence of lower, intermediate, and higher affinity ligands [J Biol Chem 263, 15392-15399 (1988)], was developed previously [J Biol Chem 265, 5055-5059 (1990)]. Such biphasic denaturation does not relate to protein domain substructure since it can occur with a protein that undergoes simple two-state denaturation in the absence of ligand. If the affinity of the protein for a ligand is great enough and if initially the protein is subsaturated with ligand, biphasic denaturation can occur. We have developed a thermodynamic description of the linkage between the equilibria for simple two-state thermal denaturation and ligand binding in which the number of cusps present in the thermogram correlates with the number of resolved steps in the plot of saturation level of remaining native protein vs. temperature (i.e. the thermal binding curve). The form of this thermal binding curve is a function of the number of ligand binding sites on the protein, the magnitudes of the association constants, and the total ligand and total protein concentrations. As a result, the constants, and the total ligand and total protein concentrations. As a result, the model indicates a possibility for multiphasic denaturation of a single cooperative unit. The presence of only two maxima in a thermogram for a protein with multiple sites on the native species derives from the form of the thermal binding curve, which in this case is a single step sigmoidal plot, and not from the predominant denaturation of unliganded and fully liganded native species. In addition, it is shown that, in general, the contributions from the denaturation of individual native protein species are decidedly non-two- state in character thereby indicating that simple deconvolution should not be carried out. In general, the interaction of ligand with denatured protein has little effect on the occurrence of biphasic denaturation although it may substantially modify peak shape and denaturation temperature(s).